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Biology of Reproduction 62, 1224-1230 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Articles

Activin A in JEG-3 Cells: Potential Role as an Autocrine Regulator of Steroidogenesis in Humans1

Xueying Nia, Shuang Luoa, Takashi Minegishib, and Chun Peng2,a

a Department of Biology, York University, Toronto, Ontario, Canada M3J 1P3 b Department of Obstetrics and Gynecology, Gumma University, Maebashi, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Activin A has been shown to exert several regulatory functions on human placenta. In the present study, we tested the hypothesis that activin A is an autocrine regulator of trophoblast using a choriocarcinoma cell line, JEG-3, as a model. Messenger RNAs for activin ßA subunit, activin binding protein (follistatin), and various activin receptors, including ActR-IA, ActR-IB, ActR-IIA, and ActR-IIB, were detected in JEG-3 cells by reverse transcription-polymerase chain reaction. The expression of activin A in JEG-3 cells was further confirmed by Western blot analysis using an antibody against activin ßA subunit. Using Northern blot analysis, Smad-2 and Smad-4 mRNAs were also observed in JEG-3 cells. These data suggest that JEG-3 cells produce activin A and express activin binding proteins and receptors, as well as potential downstream signals. In cultured JEG-3 cells, basal progesterone production was stimulated by activin A but inhibited by follistatin-288. Similarly, in the presence of androstenedione, estradiol production was enhanced by activin A but decreased by follistatin-288. On the other hand, neither activin A nor follistatin affected JEG-3 cell growth. Taken together, these findings strongly suggest that activin A is an autocrine factor that is involved in the regulation of progesterone and estradiol production in JEG-3 cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Activin, a member of the transforming growth factor ß (TGFß) superfamily, is a dimeric protein consisting of two inhibin ß subunits [1–4]. Several forms of activin have been identified in vivo including activin A: ßAßA, activin B: ßBßB, and activin AB: ßAßB [1–4]. Activin plays a critical role in reproduction. In addition to regulating pituitary FSH secretion [1–4] and gonadal functions [5–8], activin is also an important regulator of human placenta [9]. Activin A is produced by the placenta throughout pregnancy [8–10] and has been shown to stimulate production of several placental hormones, such as GnRH, hCG, inhibin, and progesterone [11, 12]. Furthermore, activin A has also been shown to regulate cytotrophoblast cell differentiation [13].

Follistatin is an activin-binding protein that neutralizes the biological effects of activin [5–8]. Similar to activin, follistatin was initially isolated from follicular fluid [14] but is now found to be produced by many tissues and cells, including the placenta [15, 16]. Alternative splicing of the follistatin gene generates two transcripts encoding follistatin-288 and follistatin-315 [17].

Receptors for activin and other members of the TGFß superfamily belong to the serine/threonine kinase receptor family [5, 18, 19]. The functional activin receptor complex consists of two types of receptors: type I and type II [5, 18, 19]. Two subtypes of the activin type II receptor, ActR-IIA and ActR-IIB, have been identified in several species, including the human [20, 21]. Similarly, two subtypes of the activin type I receptor, ActR-IA and ActR-IB [22–24], have been cloned from human tissues. Activin first binds to the type II receptor, and then this complex recruits the type I receptor. The type II receptor subsequently phosphorylates the type I receptor, which in turn phosphorylates downstream signals [25]. A novel family of proteins, termed Smads, has been identified as the intracellular signaling molecules for the TGFß family [25–27]. Several Smads, such as Smad-2, Smad-3, and Smad-4, have been shown to be involved in activin-regulated gene expression in several types of cells [26–29]. Signal transduction of activin in placental cells has not been studied.

JEG-3 cells (a choriocarcinoma cell line) have been widely used as a trophoblast cell model to study placental function [30]. JEG-3 cells produce many peptides and steroid hormones found in normal trophoblast cells, such as hCG, GnRH, and progesterone [31]. As the first step to further investigate the role of activin during pregnancy and to examine the mechanism of activin actions in human placenta, we have used JEG-3 cells as a model to examine the presence of activin, activin binding protein, activin receptors, and potential intracellular signaling molecules, and to study the effects of activin A on steroid hormone production and cell growth. The use of JEG-3 cells can avoid the contamination of other cells and tissues, such as decidual tissues, fetal membranes, and blood cells, all of which also express activin receptors [10, 21, 32].


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture

JEG-3 cells, obtained from American Type Culture Collection (Rockville, MD), were cultured in Minimal Essential Medium (MEM) supplemented with 10% fetal calf serum (FCS) and 100 U/ml penicillin-streptomycin (all reagents were purchased from Canadian Life Technologies, Burlington, ON, Canada). All cultures were maintained at 37°C in 95% air:5% CO2. Culture medium was refreshed once every 2–3 days.

RNA Isolation and Reverse Transcription-Polymerase Chain Reaction (RT-PCR)

Total RNA was isolated from JEG-3 cells using the Trizol Reagent (Canadian Life Technologies) and quantitated by absorbency at 260 nm. Two micrograms of total RNA was reverse-transcribed using the First Strand cDNA Synthesis Kit and oligo-dT12–18 primers (Amersham Pharmacia, Oakville, ON, Canada) as previously described [33]. PCR was performed in the presence of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2.0 mM MgCl2, 50 µM dNTPs, 1 U Taq DNA polymerase (Canadian Life Technologies), and specific 5'- and 3'-primers for activin ßA subunit, follistatin, or various activin receptors (see Table 1 for primer sequences) in the GeneAmp PCR system 2400 (Perkin Elmer, Foster City, CA). The PCR profile included an initial denaturation at 94°C for 90 sec, followed by 35 cycles of 94°C for 20 sec, 55–60°C (depending on the primer set) for 30 sec, and 72°C for 50 sec, and a final extension of 7 min at 72°C. For each PCR, a negative control in which the cDNA sample was replaced by water was included to test the possibility of cross-contamination. All primer sets were designed to span introns to rule out genomic DNA contamination.


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  		TABLE 1. Sequences of PCR primers

Northern Blot Analysis

Smad-2 and Smad-4 cDNA clones were isolated by screening a placental cDNA library (unpublished results). Total RNA (20 µg) were size-fractionated in a 1% agarose gel and then transferred to Hybond-N (Amersham) nylon membranes. Hybridization with 32P-labeled cDNA probes for Smad-2 or Smad-4 was carried out overnight at 42°C in 5-strength SSC (single-strength SSC is 0.15 M sodium chloride, 0.015 M sodium citrate), 5-strength Denhardt's solution, 0.5% SDS, and 50% formamide. Membranes were washed under high-stringency conditions (final wash in 0.1-strength SSC and 0.1% SDS at 65°C for 30 min). Autoradiography was performed with Kodak XAR-5 film (Eastman Kodak, Rochester, NY) and intensifying screens at -80°C for 6 days.

Protein Extraction and Western Blot Analysis

JEG-3 cells cultured in 100-mm dishes were washed with PBS, lysed with 100 µl SDS protein loading buffer (70 mM Tris-HCl, 33 mM NaCl, 1 mM Na2 EDTA, 2% SDS, 1 mM NaN3, 0.01% phenol red, and 10% glycerol; (New England BioLabs, Mississauga, ON, Canada), and scraped off the plates. The extracts were then collected and centrifuged at 12 000 x g for 5 min. Concentrations of solubilized proteins were determined using the Bio-Rad Protein Assay Kit (Bio-Rad Labs., Richmond, CA). Proteins were subsequently subjected to electrophoresis in 9% SDS-polyacrylamide gels and transferred to a polyvinylidene fluoride (PVDF) membrane (Amersham). After incubation with the blocking solution (Tris-buffered saline containing 0.1% Tween-20 and 5% skim milk) overnight at 4°C, the membrane was incubated with a monoclonal mouse anti-human activin ßA antibody (Cedarlane Laboratories Ltd., Hornby, ON, Canada) at a 1:100 dilution overnight at 4°C. Subsequently, the membrane was washed and then incubated with horseradish peroxidase-conjugated anti-mouse IgG antibody (Amersham) diluted 1:3000 for 1 h at room temperature. Immunoreactive signals were visualized with enhanced chemiluminescence reagent using the ECL detection kit (Amersham).

Activin and Follistatin Treatment of JEG-3 Cells

JEG-3 cells were seeded in 24-well plates in MEM + 10% FCS at a density of 0.5–1 x 105 cells/well. After incubation for 24 h, medium was replaced with serum-free MEM, and cells were cultured for another 24 h. Cells were then incubated with serum-free MEM (control) or serum-free MEM containing recombinant human (rh) activin A (kindly provided by Dr. A.F. Parlow, National Hormone and Pituitary Program, Rockville, MD) or rh follistatin-288 (obtained from Dr. Parlow). For experiments involving estradiol production, androstenedione (200 nM; Sigma-Aldrich Canada Ltd., Oakville, ON, Canada) was added to the culture medium as a substrate for the aromatase. For the time-course experiments, cells were treated with 30 ng/ml activin A for 2, 4, and 6 days. The culture media, including activin A, were refreshed every 2 days. At the end of the experiment, culture media were collected and cells were trypsinized. Cell numbers were determined using a hemacytometer.

RIAs

Progesterone and estradiol concentrations in culture media were determined by RIAs using [3H]progesterone and [3H]estradiol (Amersham) as tracers, respectively. Antisera for progesterone and estradiol were purchased from Sigma, and RIAs were carried out using the manufacturer's suggested procedures. The sensitivities of the RIAs were 312 pg/ml for progesterone and 100 pg/ml for estradiol. The interassay coefficients of variation were 10.5% and 9.2% for progesterone and estradiol, respectively.

Statistical Analysis

All values are expressed as the mean ± SEM of replicate cultures from a single experiment. Each experiment was repeated 2–4 times. Multiple group comparison was performed by one-way ANOVA, followed by Scheffe's multiple-comparison procedure, using the Statistical Analysis Systems (SAS) program. An unpaired Student's t-test was used if only two groups were to be compared. P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of Activin and Follistatin in JEG-3 Cells

To examine the expression of activin A and follistatin in JEG-3 cells, primers specific for activin ßA subunit were used in PCR to amplify cDNA prepared from JEG-3 cells. A DNA fragment of the expected size was observed (Fig. 1A). The PCR product was purified, subjected to direct sequencing, and found to be identical to the published human activin ßA subunit [34] (data not shown). Using a monoclonal antibody against the activin ßA subunit, Western blot analysis detected two bands with molecular sizes of 32 and 26 kDa, which correspond to inhibin A and activin A, respectively (Fig. 1B). In addition, several forms of higher molecular weight, most likely representing the precursors for ßA, were also detected (Fig. 1B). PCR using specific primers derived from human follistatin cDNA generated two DNA fragments, with sizes corresponding to follistatin-288 and follistatin-315, respectively (Fig. 1C). The primers for follistatin have been previously used to study follistatin in the human ovary, and the PCR products have been proven to be authentic [35].



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  		FIG. 1. Detection of activin ßA mRNA (A), activin A and inhibin A protein (B), follistatin mRNA (C), activin receptor mRNAs (D), and Smad mRNAs (E) in JEG-3 cells. A) RT-PCR detection of activin ßA mRNA. A DNA fragment of the expected size was observed after 35 cycles of PCR using primers derived from human activin ßA subunit cDNA sequence. B) Detection of activin A and inhibin A in JEG-3 cells. Proteins were extracted from JEG-3 cells and analyzed by Western blot using a monoclonal antibody against activin/inhibin ßA subunit. Bands of 32-kDa and 26-kDa, similar to the molecular sizes of inhibin A and activin A, respectively, were detected. C) RT-PCR analysis of follistatin mRNAs. PCR using primers specific for both follistatin-315 and follistatin-288 was performed using cDNA prepared from JEG-3 cells as the template. Two DNA fragments, corresponding to follistatin-288 and-315, respectively, were obtained. D) Detection of various activin receptor mRNAs. RT-PCR was performed on total RNA of JEG-3 cells using specific primers of activin receptors, including ActR-IA, ActR-IB, ActR-IIA, and ActR-IIB. PCR products with the expected sizes were obtained. E) Northern blot analysis of Smad-2 and Smad-4 mRNAs. Total RNA from JEG-3 cells was electrophoresed, transferred to nylon membranes, and hybridized with specific Smad-2 or Smad-4 cDNA probes, respectively. Two transcripts for Smad-2 and one transcript for Smad-4 were detected. MW, Molecular weight; C, negative control; J, JEG-3 cells

Expression of mRNA for Activin Receptors, Smad-2, and Smad-4 in JEG-3 Cells

Primers specific for various activin receptors, including ActR-IA, ActR-IB, ActR-IIA, and ActR-IIB, were used in PCR to amplify cDNA prepared from JEG-3 cells. RT-PCR resulted in the generation of DNA fragments with the expected sizes (Fig. 1D). We have previously used these primers to study activin receptor mRNA expression in granulosa-luteal cells and trophoblast cells. The generated PCR products have been characterized by Southern blot analysis, as well as by direct sequencing [36].

Smad-2 and Smad-4 have been suggested to be the downstream signals of activin receptors. To determine whether mRNAs for Smad-2 and Smad-4 are expressed in JEG-3 cells, Northern blot hybridization was performed on total RNA isolated from JEG-3 cells. Two transcripts with sizes of 4.0 kilobases (kb) and 2.9 kb were observed when the Smad-2 cDNA probe was used (Fig. 1E). One transcript with the size of 4.5 kb for Smad-4 was detected (Fig. 1E).

Effects of Activin A and Follistatin-288 on Progesterone Production by JEG-3 Cells

To determine the effects of activin on progesterone production, JEG-3 cells were treated with various concentrations of activin A for 48 h (Fig. 2A). With increasing concentrations of activin A, progesterone levels were also increased. On the other hand, treatment with follistatin-288 for 48 h dose-dependently inhibited progesterone production (Fig. 2B). Basal progesterone level decreased to 50% of the control value in cells treated with 100 ng/ml folli-statin-288.



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  		FIG. 2. Effects of activin A and follistatin-288 on progesterone production. JEG-3 cells were treated for 48 h with either serum-free MEM (control) or MEM supplemented with various concentrations of human activin A (A) or follistatin (FS)-288 (B). Each bar represents the mean ± SEM (n = 6). Different letters denote statistical difference (P < 0.05)

Effects of Activin A and Follistatin-288 on Estradiol Production by JEG-3 Cells

Similar to normal trophoblast cells, JEG-3 cells produce estradiol when an androgen is added to the culture medium [31]. To investigate the role of activin on estradiol production, JEG-3 cells were cultured with activin A or follistatin-288, in the presence of androstenedione. While activin A increased estradiol production in a dose-dependent manner (Fig. 3A), follistatin-288, at 100 ng/ml, significantly inhibited the production of estradiol (Fig. 3B).



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  		FIG. 3. Effects of activin A (A) and follistatin (FS)-288 (B) on estradiol production. Cells were incubated with serum-free MEM (control), different concentrations of activin A, or 100 ng/ml follistatin-288 in the presence of androstenedione (200 nM) for 48 h. Each bar represents means ± SEM (n = 4). Different letters denote statistical difference (P < 0.05). *P < 0.05 vs. control

Effects of Activin A and Follistatin-288 on JEG-3 Cell Growth

The effects of activin A and follistatin-288 on JEG-3 cell growth were also investigated by determining cell numbers. No significant changes in cell numbers between the control and activin A-treated groups (Fig. 4A) or between the control and follistatin-288-treated groups (Fig. 4B) were observed over the period of 48 h.



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  		FIG. 4. Effects of activin A (A) and follistatin (FS)-288 (B) on JEG-3 cell growth. Cells were incubated without (control) or with activin A or follistatin-288 for 48 h. Each bar represents means ± SEM (n = 6)

Time-Course Effect of Activin A

Cells were cultured with control medium or 30 ng/ml of activin A for 2, 4, and 6 days. Activin A significantly increased progesterone (Fig. 5A) and estradiol (Fig. 5B) production at Day 2 and Day 4. However, after 6 days of treatment, there were no significant differences in either progesterone or estradiol levels between the control and the activin A-treated group (Fig. 5, A and B). The cell number increased as incubation time increased. However, there was no significant difference between the control and activin A-treated group at any of the time points tested (Fig. 5C).



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  		FIG. 5. Time-course effect of activin A on the production of progesterone (A), estradiol (B), and cell growth (C). Cells were cultured with serum-free medium (control) or medium containing 30 ng/ml of activin A, in the presence of 200 nM androstenedione, for 2, 4, and 6 days. Culture medium, including activin A, was refreshed every 2 days. Each bar represents means ± SEM (n = 4). *P < 0.05 vs. control


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study demonstrates that mRNAs for the activin ßA subunit, activin receptors, and proposed activin downstream signals, as well as activin A protein, are expressed in JEG-3 cells. In addition, activin A stimulates, while follistatin-288 inhibits, progesterone and estradiol production by JEG-3 cells. Since follistatin is generally considered to be a binding protein whose role is to neutralize activin bioactivity [5, 8, 37], the inhibitory effect of follistatin on progesterone and estradiol production by JEG-3 cells indicates that activin A produced by JEG-3 cells contributes to the basal steroid production. Thus, activin A appears to play an autocrine role in regulating steroidogenesis in JEG-3 cells.

Using RT-PCR, mRNAs for activin ßA subunit and activin receptors were detected in JEG-3 cells. The authenticity of the PCR product of the activin ßA subunit was confirmed by sequencing. The primers designed to examine activin receptor mRNA expression have been used in our previous study on normal human granulosa-luteal and trophoblast cells [36], and the generated DNA fragments were confirmed to be identical to the respective published sequences [20–23]. Since all primer sets span introns, the possibility of genomic DNA contamination can be ruled out. Western blot analysis using a specific antibody for ßA subunit demonstrated that activin A is present in JEG-3 cells. These results suggest that JEG-3 cells produce activin A and may express various activin receptors. The production of activin A by normal trophoblast cells has been demonstrated [16]. Our previous studies have shown that trophoblast cells from first-trimester and term placentas express mRNAs for all four known subtypes of activin receptors [33, 36]. It is therefore probable that JEG-3 cells retain the ability to produce activin and activin receptors as do normal trophoblast cells.

Smad-2 and Smad-4 have been shown to mediate transcriptional activity of activin and TGFß [26, 29]. Smad-2 can be phosphorylated by type I receptors of activin and TGFß, whereas Smad-4 is a general downstream partner to translocate Smads into nuclei [25, 38]. Northern blot analysis detected Smad-2 and Smad-4 mRNA expression in JEG-3 cells. The sizes of Smad-2 and Smad-4 mRNAs observed in JEG-3 cells are similar to those found in other tissues [39, 40], suggesting that no deletion of Smad-2 or Smad-4 mRNA has occurred in JEG-3 cells. In several tumor tissues or cell lines, such as pancreatic, breast, ovary, head, neck, and esophagus cancers, mutations on the Smad-4 gene have been described [25, 41]. Smad-2 mutations have also been detected in colon, head, and neck cancers [42, 43]. Our preliminary study has revealed that there is no mutation on the Smad-4 mRNA sequence (unpublished results). Whether or not Smad-2 and Smad-4 mediate activin A-induced steroid hormone production remains to be investigated.

In the present study, we observed that activin A stimulates progesterone and estradiol production by JEG-3 cells in a dose-dependent manner. These results are consistent with the stimulatory effect of activin A on progesterone production by both first-trimester and term placentas [9–11, 15]. The stimulation of estradiol production by activin A is also in agreement with the reported action of activin on aromatase activity in human trophoblast cells [44]. The effective doses of activin A on JEG-3 cells are similar to those found in normal trophoblast cells [11, 12, 15]. Our results also demonstrate that the effects of activin on steroid production are time-dependent. Treatment with activin A for 2 or 4 days significantly increased the production of both progesterone and estradiol. However, after 6 days of treatment, no significant difference in progesterone or estradiol levels between the control and activin A-treated groups was found. One possible explanation is that the cell density is too high after 6-day culture. When cell number reaches 3–4 x 105/well, cells grow in multiple layers, and this may have limited the exposure of cell surfaces to activin A. To explore this possibility, we examined the relationship between the cell density and the progesterone responsiveness to activin A. The optimal response of progesterone production to activin A was observed when cell density was 1–1.5 x 105/well. When cell density reached 3 x 105/well, no significant response to activin A was found (data not shown).

Follistatin binds to activin with high affinity, with Kd values similar to those of activin receptors [5, 45]. Two mechanisms by which follistatin neutralizes activin bioactivity have been proposed. One is by inhibiting the binding of activin to its type II receptors [37], and the other is by increasing the endocytotic degradation of activin through the association of follistatin-288 with cell surface heparan sulfate [46]. Petraglia et al. [32] reported that human placenta expressed follistatin mRNA and protein, and that follistatin-288 blocked activin-induced progesterone production by trophoblast cells [15]. In the present study, we demonstrated that follistatin-288 significantly inhibited basal progesterone and estradiol production. Follistatin has been shown to bind to inhibin, but with a lower affinity than activin [5, 45]. Although we showed that JEG-3 cells also produce inhibin A, the reduced production of steroids by JEG-3 cells after follistatin-288 treatment is unlikely to have resulted from the interaction between follistatin and inhibin, as our preliminary data indicate that inhibin A inhibits basal progesterone production (unpublished data). Thus, the findings from this study support the notion that activin A is produced by JEG-3 cells and, in turn, stimulates steroidogenesis of JEG-3 cells.

Activin A has been shown to regulate cell proliferation. Depending on specific cell types, both inhibitory [47–49] and stimulatory [50] effects have been reported. In the present study, we also determined whether activin A affects JEG-3 cell growth. Over a 2-day or a 6-day culture period, activin A did not affect JEG-3 cell numbers. Similarly, follistatin-288 also had no effect on JEG-3 cell growth. These results indicate that activin is not a regulator of JEG-3 cell growth and that effects of activin and follistatin on steroid hormone production were not the result of changes in cell numbers. In preliminary experiments, we also found that treatment with activin A had no effect on cell growth of normal first-trimester trophoblast cells (unpublished results). Although activin A stimulates trophoblast cell differentiation [13], our data suggest that it does not regulate trophoblast cell proliferation.

In summary, the present study demonstrates that JEG-3 cells may produce activin A and follistatin, and express mRNA of all known activin receptors and their potential downstream signaling molecules. Activin A has stimulatory effects on progesterone and estradiol production by JEG-3 cells, whereas follistatin inhibits steroidogenesis. These findings strongly suggest that activin A is an autocrine factor that regulates steroid hormone production in JEG-3 cells. In addition, our data also indicate that the JEG-3 cell line is a suitable model for further study of the function and signal transduction of activin A in the human placenta.


   ACKNOWLEDGMENTS
 
We thank the National Hormone and Pituitary Program and Dr. A.F. Parlow for providing recombinant human activin A and follistatin-288.


   FOOTNOTES
 
First decision: 10 August 1999.

1 This study was supported by a grant from Medical Research Council of Canada to C.P. C.P. is a recipient of Women's Faculty Awards from the Natural Sciences and Engineering Research Council of Canada. Some preliminary results have been presented as a poster at the 80th Annual Meeting of the Endocrine Society; June 23–27, 1998; New Orleans, LA. Back

2 Correspondence: Chun Peng, Department of Biology, York University, 4700 Keele St., Toronto, ON, Canada M3J 1P3. FAX: 416 736 5698; cpeng{at}yorku.ca Back

Accepted: December 6, 1999.

Received: June 23, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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